1. Field of the Invention
The present invention relates to immunoassays useful for distinguishing between the insect pests Heliothis virescens and Helicoverpa zea. More particularly, these immunoassays employ either Heliothis virescens-specific antibodies which do not detectably cross-react with eggs of Helicoverpa zea, or Helicoverpa zea-specific monoclonal antibodies which do not detectably cross-react with eggs of Heliothis virescens.
2. Description of the Related Art
Heliothis virescens (also known as the tobacco budworm) and Helicoverpa zea (also known as the cotton bollworm or corn earworm) both have the capacity to inflict devastating yield losses to agronomically important crops. These two insect pests are typically controlled through the use of pyrethroid and Bacillus thuringiensis (Bt) insecticides. However, Heliothis virescens has the tendency to become resistant to pyrethroids, while such resistance has not been observed in Helicoverpa zea. Conversely, Helicoverpa Zea exhibits tolerance to Bt, which does not occur in Heliothis virescens.
In order to retard the development of pyrethroid resistance in Heliothis virescens, current control methods are aimed at preventing the overexposure of populations of Heliothis virescens to pyrethroids. Such control methods would be facilitated if Heliolthis virescens and Helicoverpa zea populations could be conclusively distinguished in the field at an early stage, e.g., the egg stage. Immunoassays have been used in the past to distinguish between Heliothis virescens and Helicoverpa zea populations. However, in these immunoassays, antibodies were employed which exhibited at least some degree of cross-reactivity, i.e., the Helicoverpa zea-specific antibodies employed cross-reacted with Heliothis virescens eggs. Thus, these prior art methods, which are summarized below, were unable to conclusively distinguish between Heliothis virescens and Heiicoverpa zea organisms.
Trowell et al 1993 (In Pest Control and Sustainable Agriculture, ed. S. Corey, D. Dall, W. Milne, pp. 176-179, Melbourne: CSIRO Press) discloses two monoclonal antibodies, designated 70.5.31.11 and 70.7.88.1, which recognize Helicoverpa armigera for use in assays to distinguish between Helicoverpa armigera and Helicoverpa punctigerca. Trowell et al 1993 states that "[w]e believe it will be possible to modify the test kit to allow discrimination of two economically important North American heliothine species, Heliothis virescens and Helicoverpa (Heliothis) zea . . . "(p. 178, 2.sup.nd col., 2.sup.nd full paragraph). However, no evidence is given that these monoclonal antibodies react specifically (i.e., with no detectable cross-reactivity) with either Heliothis virescens or Helicoverpa zea, or that they react with either of these species at all.
Trowell et al 1994 (Canadian Patent Application 2,114,160) discloses monoclonal antibodies 70.5.31.11 and 70.7.88.1 described above, as well as monoclonal antibody 21.91.2. Trowell et al 1994 states that the antibodies of their invention are "capable of binding a target molecule of H. zea but not Heliothis virescens." (page 7, lines 32-33). Trowel et al 1994 also states that "[t]he antibody [21.91.2] recognised. H. zea but not Heliothis virescens . . . " (page 25, lines 6-8).
However, as in Trowell et al 1993, no evidence is given that monoclonal antibodies 70.5.31.11 and 70.7.88.1 react specifically with either Heliothis virescens or Helicoverpa zea, or that they react with either of these species at all. Moreover, Trowell et al 1994 states that "[a] second batch of supernatant from 21.91.2 was tested and, in this experiment, the inmnunoreactivity appears to be dependent on the concentration of egg proteins (FIG. 1b). The best discrimination was observed with dilutions of H. armigera and Heliothis virescens egg proteins of 1/32 or 1/64." (page 25, lines 11-15). Thus, it appears that monoclonal antibody 21.91.2 does cross-react with eggs of Heliothis virescens.
Greenstone et al 1989 (Ann. Entomol. Soc. Am. 82:45-49); Stuart et al 1990 (Arm. Entomol. Soc. Am. 83:1101-1107); and Greenstone 1993 (Entomol. Exp. Appl. 68:1-7) disclose the use of immunological assays (i.e., an immunodot assay and an enzyme4 inked immunosorbent assay (ELISA)) to analyze the contents of the stomachs of arthropod predators in predator-prey studies. These assays employ monoclonal antibody HZ5-1 which is specific for Heiicoverpa zea arylphorin. HZ5-1 was prepared using hemolymph of fifth instars of Heiicoverpa zea. Greenstone et al 1989 states that HZ5-1 "distinguishes remains of [Helicoverpa zea] fifth instars in enzyme-linked immunosorbelt assay (ELISA) of fed predator extracts from the remains of the all other instars and eggs of this and related species [e.g., Heliothis virescens]." (abstract). Stuart etal 1990 employs HZ5-1 in an immunodot assay and states that "[a] remarkable aspect of our results is the complete lack of overlap in color development between known positives [e.g., fifth inslar Heiicoverpa zea] and negatives [e.g., Heliothis virescens]--not even a very faint dot is formed in negative assays." (p. 1104, col. 2). However, Greenstone et al 1993 states that "[o]ngoing research utilizing different membranes and other substrates indicates that background can be entirely eliminated, enhancing the distinction between positive and negative results . . . " (p. 5, col. 2 to p. 6, col.1).
Thus, it appears that HZ5-1 recognizes remains of Heiicoverpa zea fifth instar larvae, and does not detectably react with Heliothis virescens, only if immunoassay conditions are manipulated to eliminate background. In other words, lack of crossreactivity is not an inherent characteristic of HZ5-1, but rather depends on the immunoassay conditions employed. Moreover, in contrast to the monoclonal antibodies of the present invention, HZ5-1 does not react with egg proteins, and therefore cannot be used to identify insects at the egg stage.
Greenstone et al 1995 (J. Econ. Entomol. 88:213-218) describes monoclonal antibody HZE-1 for use in a squashblot immunoassay to distinguish Heiicoverpa zea eggs, from Heliothis virescens eggs. HZE-1 was prepared using Heiicoverpa zea whole egg homogenate as immunogen. Although HZE-1 is specific for Heiicoverpa zea eggs, is Greenstone et al 1995 states that "H. virescens eggs sometimes appeared weakly positive when tested on membranes blocked with phenylhydrazine or peroxidase inhibitor . . . " (p. 214, col. 2, 3.sup.rd full paragraph).
Goodman etal 1997 (Ann. Entomol. Soc. Am. 90:83-90) discloses and characterizes monoclonal antibodies HZE-1 and HVE-1 for use in ecological studies of predation. HVE-1 was prepared using purified Heliothis virescens vitellin as inmuunogen. Both antibodies were shown to recognize vitellin specifically. However, HZE-1 recognizes Helicoverpa armigera vitellin and Helicoverpa punctigera vitellin in addition to Heiicoverpa zea vitellin. Furthermore, HVE-1 recognizes vitellins of a number of Helicoverpa and Heliothis species (including Heiicoverpa zea), and therefore is not specific for Heliothis virescens vitellin only.
Greenstone et al 1997 (U.S. Pat. No. 5,656,437) discloses monoclonal antibody HZE-1. The inventor states that he has "now discovered a hybridoma cell line which produces and secretes a monoclonal antibody which specifically binds to vitellin in the eggs of the corn earworm, Heiicoverpa zea, but does not bind to vitellin in the eggs of the tobacco budworm, Heliothis virescens." (col. 2, 11. 43-47). Of course, this statement is contradicted by the statement in Greenstone etal 1995 that "H. virescens eggs sometimes appeared weakly positive" in an immunoassay employing HZE-1.
Greenstone et al 1997 (U.S. Pat. No. 5,656,437) used only one antibody specific to Heiicoverpa zea eggs. Using a single antibody to identify two morphologically similar species can result in false negatives if the eggs are not those of Heiicoverpa zea. In the field, eggs of several insect species visually resemble those of Heiicoverpa zea and Heliothis virescens, increasing the likelihood of obtaining false negative results with just one antibody.